ABSTRACT The Kv3.3 channel is the major ?high threshold? voltage-dependent potassium channel in the auditory brainstem. Mutations in Kv3.3 severely impair the ability of humans to localize sounds in space, can produce tinnitus, and also lead to neuronal degeneration, including loss of cerebellar neurons. The Kv3.3 channel differs from all other members of the Kv3 family of channels in that it has an extended cytoplasmic C-terminus. This contains a proline-rich domain conserved in proteins that activate actin nucleation through the Arp2/3 complex. The association of Kv3.3 with Arp2/3 is required for normal channel function, and when this is disrupted, the channel rapidly inactivates. The cytoplasmic C-terminal domain also directly binds the Arp2/3- activating protein Hax-1, and caspase-7, both of which regulate neuronal survival. Experiments in this proposal will test the role of Kv3.3-Arp2/3 and Kv3.3-Hax-1 interactions by testing the effects of disruption of these interactions on the properties of auditory brainstem neurons that express Kv3.3 at high levels, including the calyx of Held presynaptic terminal within the medial nucleus of the trapezoid body. Patch clamp recordings and immunocytochemical studies will be carried out to examine intrinsic excitability, synaptic transmission and morphology in response to treatments that disrupt these interactions in wild-type mice. These results will then be compared with those obtained in Kv3.3-/- animals and knock-in mice bearing a specific human Kv3.3 mutation (G592R) that does not impede channel function but disrupts Kv3.3-cytoskeletal interaction. We will determine whether the G592R mutation produces any degenerative changes in auditory nuclei, as is known to be the case for cerebellar neurons in patients with this mutation. In vitro studies and experiments with transfected cells will define the specific domains in Kv3.3 and Hax-1 required for protein-protein interactions. Finally, we will test the actions of a novel class of compounds that act on Kv3 channels to determine whether they alter the effects of the human G592R mutation. Our findings suggest that Kv3.3 channels may be a new therapeutic target for hearing disorders such as tinnitus and other disorders of hearing that occur during aging, potentially linked to the degeneration of subsets of neurons or their synaptic connections.